title: Residue substitutions near the redox center of Bacillus subtilis Spx affect RNA polymerase interaction, redox control, and Spx-DNA contact at a conserved cis-acting element.
abstract: Spx, a member of the ArsC protein family, is a regulatory factor that interacts with RNA polymerase (RNAP). It is highly conserved in Gram-positive bacteria and controls transcription on a genome-wide scale in response to oxidative stress. The structural requirements for RNAP interaction and promoter DNA recognition by Spx were examined through mutational analysis. Residues near the CxxC redox disulfide center of Spx functioned in RNAP α subunit interaction and in promoter DNA binding. R60E and C10A mutants were shown previously to confer defects in transcriptional activation, but both were able to interact with RNAP. R92, which is conserved in ArsC-family proteins, is likely involved in redox control of Spx, as the C10A mutation, which blocks disulfide formation, was epistatic to the R92A mutation. The R91A mutation reduced transcriptional activation and repression, suggesting a defect in RNAP interaction, which was confirmed by interaction assays using an epitope-tagged mutant protein. Protein-DNA cross-linking detected contact between RNAP-bound Spx and the AGCA element at −44 that is conserved in Spx-controlled genes. This interaction caused repositioning of the RNAP σA subunit from a −35-like element upstream of the trxB (thioredoxin reductase) promoter to positions −36 and −11 of the core promoter. The study shows that RNAP-bound Spx contacts a conserved upstream promoter sequence element when bound to RNAP.
authors.affiliation: Division of Environmental & Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University, Beaverton, Oregon, USA.
authors.forename: Ann A
authors.initials: AA
authors.lastname: Lin
authors.forename: Don
authors.initials: D
authors.lastname: Walthers
authors.forename: Peter
authors.initials: P
authors.lastname: Zuber
mesh_terms: D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D050505:Mutant Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases